*Purpose: Despite profound advances in immunosuppression, intestinal transplantation (ITx) is plagued by frequent cellular rejection. T regulatory cells (Tregs) are key in mitigating immune cell activation but their phenotype and function in ITx is unknown.

*Methods: Samples were taken from 34 healthy ITx patients and 23 patients with a history of rejection both from our IRB-approved Immunomonitoring and Tissue Bank Study. Polychromatic flow cytometry (PFC) was used to analyze blood and intestinal allograft samples to characterize surface receptor phenotype, transcription factor expression, and cytokine production. Demethylation pyrosequencing of the Treg-specific demethylated region (TSDR), co-culture suppressive assays with Tregs and responder T cells (Tresp), and RNAseq were also conducted.

*Results: Biopsy PFC showed significantly more CCR6+CXCR3+ chemokine receptor expressing Treg cells in rejection patients compared with ITx controls. They expressed more Th17 transcription factor ROR-γt and produced increased IL-17 and IFN-γ. Functionally there was similar FoxP3 TSDR demethylation in both groups, and Tregs in rejection patients continued to suppress T responder cell proliferation. RNAseq analysis revealed upregulation of classic Treg genes such as FoxP3, TIGIT, and ENTPD1 as well as key genes involved in purinergic signaling including ADORA2A, DPP4, and LRRC32 in rejection samples, highlighting an escalated level of activation during rejection.

*Conclusions: Our research demonstrates that Th17 polarized Treg cells, which are critical in inflammatory conditions, are present during ITx rejection and appear to retain their normal function. Further study could elucidate the therapeutic potential of these cells in treating ITx rejection.

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